Cytochromes are heme proteins essential for the aerobic and anaerobic growth of most organisms, including human pathogens. Relatively recently it has become clear that dedicated assembly pathways and factors are crucial for cytochrome biogenesis. The assembly of c-type cytochromes occurs by one of three pathways, called systems I, II, and III. Systems I and II have nine and four assembly factors (membrane proteins) respectively, while system III uses a single enzyme called cytochrome c heme lyase. Defects in system III result in certain human genetic diseases. Because only prokaryotes, plants, and protozoa use systems I or II, these pathways represent potential targets for antimicrobial agents. The c-type cytochromes possess heme that is covalently ligated to the apocytochrome at two cysteines, which must be reduced for attachment to the heme. Since assembly of c-type cytochromes occurs at the outer surface of the inner membrane, cells must deliver reduced heme, synthesized inside the cell, to the secreted unfolded apocytochrome. In this application we take advantage of our previous success for reconstituting all three systems in recombinant Escherichia coli to study the in vivo concentrations and features of heme that are optimal for each system. The heme delivery pathways will be established. Furthermore, the protein(s) required for the final cytochrome c synthetase (ligase) activity of each system will be purified and characterized for heme and apocytochrome c ligation properties, binding, and mechanisms. Based on our findings that certain metal porphyrins are specific inhibitors of the systems I and II pathways, other porphyrin analogs will be tested for their ability to be ligated to apocytochrome c (or to inhibit biogenesis), further delineating the properties of heme that are critical for ligation. In summary, this study will address which protein(s) constitute the cytochrome c synthetases, how heme is delivered, what concentrations and features of heme are required for each system. Results will help unravel why three very different pathways have emerged in nature and mechanisms to target them.